Background There are five different anti-TNF biologics: three are bivalent full length (FL) antibodies (adalimumab, golimumab, and infliximab), one a bivalent fusion protein (etanercept), and one a univalent PEGylated Fab9 (PF) (certolizumab pegol [CZP]). Administration of such protein biologics can induce anti-drug antibodies (ADAbs), of which the majority are anti-idiotypic antibodies (anti-ID).1 The potential cross-linking of bivalent anti-IDs with bivalent biologics can result in the formation of large immune complexes (ICs), which are subsequently cleared by mechanisms using the multiple Fc domains, thereby resulting in a decrease in the efficacy of the biologic. Since univalent biologics, such as CZP, only have one binding Fab9 arm, such large cross-linked anti-ID-mediated ICs are unlikely to form. Therefore, anti-IDs may have a different effect on the elimination and bioavailability of univalent and bivalent biologics in vivo. Objectives To determine if the valency of a biologic will affect the in vitro size and in vivo elimination from the plasma of ICs formed with an anti-ID following intravenous (IV) administration to BALB/c mice. Methods An anti-ID antibody to CZP was generated and used for subsequent studies to mimic an ADAb response. Univalent PF CZP was reengineered as a bivalent FL humanized IgG1 antibody (similar to adalimumab) to directly compare the effect of valency on IC-mediated clearance. This FL antibody showed very similar TNF neutralization compared to the conventional PF molecule in a bioassay. To generate ICs, the anti-ID was incubated overnight with either PF or FL CZP, and the size of the resultant ICs determined by analytical ultracentrifugation (AU). The anti-ID complexes and PF and FL CZP alone were then administered IV to the mice, and the elimination of the anti-TNFs from the circulation was monitored by quantitative liquid chromatography-mass spectrometry (LC-MS) in serial plasma samples. Results AU analysis of the immune complexes formed between PF CZP and the anti-ID showed the presence of one peak corresponding in size to one anti-ID molecule bound to two PF molecules (∼3.5x105 Da). In contrast, the FL CZP/anti-ID mixture showed ICs of various sizes up to very large molecular weights (>1x106 Da), with the predominant species corresponding to a complex of two anti-IDs bound to two FL CZP molecules (∼6x105 Da). The in vivo studies showed that the FL CZP/anti-ID ICs were eliminated much faster (t1/2=0.3 hours) than the FL CZP alone (t1/2=44.5 hours), whereas the PF CZP/anti-ID ICs were eliminated more slowly (t1/2=60.5 hours) than the PF CZP alone (t1/2=19.7 hours). Conclusions The FL CZP molecule formed large ICs with the anti-ID, which led to much faster elimination than the FL molecule alone. This result suggests that an ADAb response to a FL antibody could lead to rapid elimination and loss of efficacy of the drug in patients. In contrast, the PF CZP/anti-ID complex had a longer half-life than the PF CZP alone, presumably because this molecule was not seen as an IC due to the presence of only one Fc. These results showed that an ADAb to a univalent biological reagent may not lead to elimination and instead, may actually increase the in vivo half-life of the molecule. References van Schouwenburg PA. Ann Rheum Dis 2013;72:104–9. Acknowledgements This study was funded by UCB Pharma. Editorial services were provided by Costello Medical Consulting. Disclosure of Interest J. Silva Employee of: UCB Pharma, A. Nesbitt Employee of: UCB Pharma

BackgroundCertolizumab pegol (CZP) is a PEGylated, Fc-free anti-TNF that lacks the Fc portion found in monoclonal antibodies. Infliximab (IFX) and adalimumab (ADA) are both antibodies, while etanercept (ETA) is a receptor fusion protein, and all three of these anti-TNFs have an IgG1 Fc. In mothers treated with CZP, it has been reported that lower levels of CZP, compared to ADA or IFX, are transferred to the neonate.1 It has been suggested this transfer differential may be due to the one-way active transport of antibodies across the placenta thought to be mediated by the neonatal Fc receptor (FcRn). However, anti-TNF binding to FcRn, and FcRn-mediated transcytosis across a cell layer, have not been studied.ObjectivesTo quantify binding of the anti-TNFs CZP, IFX, ADA and ETA to FcRn and to measure FcRn-mediated transcytosis of these agents across a cell layer.MethodsA Biacore™ assay was used to determine the binding of CZP, ADA and IFX to human FcRn. Anti-TNFs were passed over an FcRn-coated chip for 5 minutes at a range of concentrations from 21-670nM to determine the on-binding rate; a buffer at pH 6.0 was used to allow optimum binding. The off-rate was followed for a further 5 minutes by running buffer alone over the chip. MDCK II cells transfected with human FcRn were used to measure FcRn-mediated transcytosis across a cell layer using a pH 5.9 buffer on the apical side and pH 7.2 on the basolateral side. The anti-TNFs and the control antibody (P146), which possessed a Fc modified to prevent binding to FcRn, were biotinylated to allow visualization. The amount of each anti-TNF transcytosed across the cell layer over 4 hours was measured by MSD assay.ResultsIFX (132nM) and ADA (225nM) had relatively high binding affinity to FcRn while the binding affinity of ETA to FcRn was approximately 5 to 10-fold lower (1500nM), similar to previously reported results.2 In contrast, CZP did not bind to the FcRn with any measurable affinity. The levels of transcytosis seen with IFX and ADA were 249.6ng/mL and 159.5ng/mL, respectively (mean of 3 experiments), over 4 hours. Transcytosis of ETA (81.3ng/mL) was lower than that of ADA and IFX. In contrast, the level of CZP transcytosis was significantly lower, at 3.2ng/mL, than that observed with the other anti-TNFs tested. The control antibody P146 also showed a low level of transfer at 5.9ng/mL. Since neither the control antibody nor CZP bind to FcRn, the levels detected are probably due to a low level of non-specific leakage across the cell layer.ConclusionsThis is the first report to quantify the binding of anti-TNFs to FcRn and their FcRn-mediated transcytosis across a cell layer. CZP does not have an Fc and thus did not bind to FcRn. Moreover, no FcRn-mediated CZP transcytosis was detected. In contrast, ADA and IFX had a relatively high binding affinity to FcRn and were actively transcytosed across the cell layer. ETA showed lower binding affinity to FcRn and subsequent transcytosis, compared to IFX and ADA, but FcRn-mediated transport could still be measured. These results explain the previously observed active transport of anti-TNFs across the placenta seen in patients treated with IFX and ADA, whereas only low levels were observed with CZP.1ReferencesMahadevan U. Clin Gastroenterol Hepatol 2012 [epub ahead of print]; 2. Suzuki T. J Immunol 2010;184(4):1968-1976.AcknowledgementsThe authors acknowledge Costello Medical Consulting for writing and editorial assistance which was funded by UCB Pharma.Disclosure of InterestT. Baker Employee of: UCB Pharma, L. Kevorkian Employee of: UCB Pharma, A. Nesbitt Shareholder of: UCB Pharma, Employee of: UCB Pharma

Certolizumab pegol (CZP) has a different mode of action from the other anti-TNFs, infliximab (IFX), adalimumab (ADA) and etanercept (ETA), that may be due both to its structure and how it signals through membrane TNF-α (mTNF-α). Different effects of this signalling could relate to the exact epitopes to which the anti-TNFs bind. We investigated the binding epitope for CZP and how it differs from the other anti-TNFs.

Differences have been seen among the anti-TNFs in mediating reverse signalling of membrane TNF-α (mTNF-α). Natural killer (NK) cells express high levels of mTNF-α and may be involved in rheumatoid arthritis pathogenesis. We examined the effect of certolizumab pegol (CZP) and the other anti-TNFs, adalimumab (ADA), etanercept (ETA) and infliximab (IFX), on cellular activities of NK cells.

Background and objectives Certolizumab pegol (the only polyethylene glycolylated (PEG) Fab9 anti-tumour necrosis factor (CZP)) has a very low incidence of injection site pain (ISP) in clinical studies. This could be due to inhibition of mast cell degranulation by the CZP PEG component. Mast cells could be involved in mediating ISP: they are present at high numbers in the skin and can rapidly secrete inflammatory mediators in preformed granules. Changes in Ca2+ flux are primary indicators of cell activation, and mast cell degranulation is preceded by Ca2+ flux into the cell. PEG binds to metal ions (in particular Ca2+). The aim of this study was therefore to determine if PEG could inhibit Ca2+ flux in a cellular system. Materials and methods Peripheral blood monocytes, isolated using MACS beads, were incubated with the fluorophore Fluo-4. Ca2+ flux was measured using flow cytometry by detecting the Fluo-4 emission at 515–535 m. Ionomycin was added at 2 μg/ml and the emission measured over a 4 min period relative to background (assessed prior to ionomycin addition). The effect of a range of concentrations of the 40 kDa PEG component of CZP on Ca2+ flux induced by ionomycin was assessed. The effect of PEG on an ionomycin-induced Ca2+ flux in cultured mast cells was determined by a similar method but flux was measured using a fluorimeter. Results Ionomycin caused a dramatic flux of Ca2+ in the monocytes. PEG caused a dose-dependent inhibition of Ca2+ flux over a range of concentrations from a minimum of 40 mg/ml. This is a physiological concentration of PEG as the equivalent local concentration of PEG which is injected into patients is 88.9 mg/ml. The inhibitory concentration (IC50) for inhibition of the Ca2+ flux caused by PEG was around 10 mg/ml. The maximum inhibition observed was 84.2% obtained at 40 mg/ml, with the effect titrating out around 1 mg/ml. In mast cells the IC50 was around 11 mg/ml. Conclusion The PEG component of CZP inhibits Ca2+ flux in monocytes and mast cells at a concentration relevant at the site of injection. This inhibition of Ca2+ flux could potentially explain the low levels of ISP observed with CZP in the clinic. This effect would only be observed at the site of injection as systemic concentrations of the drug are below the levels where an effect is seen.

Background: Activities of the anti-TNFs, certolizumab pegol (CZP), etanercept (ETA), infliximab (IFX) and adalimumab (ADA), have been compared in a range of in vitro assays. CZP is the only licensed PEGylated Fab’ anti-TNF; ETA is a fusion protein with an IgG1 Fc, and IFX and ADA are both antibodies with an IgG1 Fc. Golimumab (GLM) is a monoclonal IgG1 TNF inhibitor recently approved for a number of indications; it is thus of interest to assess the in vitro activity of GLM. In vitro assays previously used were neutralisation of TNF in the L929 bioassay, inhibition of LPS-driven cytokine production by monocytes, induction of apoptosis in activated lymphocytes and monocytes, and induction of neutrophil necrosis. Methods: Neutralisation of human TNF was assessed in the L929 bioassay using a range of concentrations of the anti-TNFs and a fixed concentration of TNF (100 pg/mL). Activity of the anti-TNFs at inhibiting LPS-driven IL-1β secretion by monocytes was assessed by incubating peripheral blood monocytes with various concentrations of the anti-TNF for 1 hour (hr) and then washing the cells. LPS was added for 4 hrs, the supernatants collected and the IL-1β level measured by ELISA. To assess induction of apoptosis, peripheral blood lymphocytes were activated for 2 days with 2 μg/mL CD3/CD28 and monocytes with 300 U/mL IL-4 and GMCSF for 3 days. The effect of the anti-TNFs on apoptosis was assessed by Annexin V staining using flow cytometry 24 hrs later. The effect of the anti-TNFs on neutrophil necrosis was determined by measuring myeloperoxidase release after 12 hrs. An isotype-matched control was used in all assays except the L929 bioassay. Results: IC90 neutralisation activity of the anti-TNFs in the L929 bioassay was 0.3 ng/mL for ETA, 4 ng/mL for GLM, 15 ng/mL for ADA, and 20 ng/mL for IFX, compared with 2.5 ng/mL for CZP. CZP was the most potent inhibitor of LPS-driven IL-1β secretion (IC50 ∼0.1 ng/mL), followed by GLM (20 ng/mL) and IFX (50 ng/mL). GLM, ADA, IFX and ETA induced apoptosis of monocytes and lymphocytes to a similar degree reaching a level of 23% and ∼40% at 100 μg/mL, respectively. CZP caused no increase in apoptosis above the levels seen with the isotype-matched control. In the neutrophil necrosis assay, ADA,IFX and GLM caused ∼70% necrosis at 100 μg/mL, and ETA 48%. CZP did not increase the level of necrosis above the level of the control. Conclusions: Bioactivity of the IgG1 molecules GLM, IFX and ADA in neutralising human TNF was inferior to that of CZP and ETA. CZP, the only PEGylated anti-TNF, had a different profile to the other anti-TNFs as it was the most potent at inhibiting LPS-driven IL-1β production by monocytes, did not induce apoptosis of activated monocytes and lymphocytes, and did not cause neutrophil necrosis. The clinical relevance of these in vitro effects is unknown. Nevertheless, these assays show interesting in vitro differences between the anti-TNFs. Disclosure statement: G.F. and A.N. are employees of UCB.

Certolizumab pegol (CZP), a PEGylated Fab', has been shown to distribute to inflamed versus normal tissue at a ratio of 3.8 in a collagen-induced arthritis (CIA) mouse model, whereas adalimumab (ADA), an IgG, had a ratio of 1.9.1 The objective of this study was to compare PEGylated Fab' and IgG distribution by determining the consequence of tumour necrosis factor (TNF) binding on the distribution of these reagents in inflamed versus normal tissue in the …

Introduction: Ten years after approval of infliximab (IFX) for treatment of Crohn's disease (CD), time has come to analyse the long-term outcome of this revolutionary agent.Aim was to assess the long-term safety of CD patients treated with IFX in a single referral center cohort.Methods: All patients treated with IFX were assessed from our CD patients' database.Medical charts were retrospectively reviewed.Endpoints were the number of serious adverse events (SAE), number of malignancies, serious or opportunistic infections and the number of deaths.SAE was defined as any adverse event (AE) that resulted in hospitalisation or was fatal / life threatening or led to disability.Results: 430 CD patients (280 female (65.1%) received IFX.Mean age at diagnosis and at time of start with IFX were 24.0 and 33.0, respectively.Follow-up was completed in 366 patients (85%), with a median follow-up of 4.5 years (IQR 2.7 -6.7).Thirty-six SAE's occurred in 33 patients (9.0%) (see table ).Infusion reactions were noted in 64 patients (17.5%).Three patients had an infusion reaction that required hospitalisation and were therefore considered SAE.Malignancy was found in 12 patients (3.3%).Eleven patients (3.0%) experienced a serious infection requiring hospitalization.All infusion reactions (n=64) and serious infections (n=11) were considered to be probably related to IFX therapy; 4 malignancies were possibly related and also the cardiovascular and perforating SAE's were considered possibly related.8 patients died during or after IFX treatment (2.2%) at a median age of 48, after a median of 3.5 infusions.Conclusion: In our cohort, the total number of SAE was low in comparison to literature.The number of deaths and malignancies found were comparable, indicating that other adverse events may be underreported in the charts.SAE's in a cohort of 366 CD patients treated with IFX *3 patients had 2 different SAE W1205

Background and objectives Differences in structure and in vitro properties of anti-tumour necrosis factor (TNF) agents may account for different modes of action and clinical outcomes. The activities of certolizumab pegol (CZP), etanercept (ETA), infliximab (IFX) and adalimumab (ADA) have been compared using in vitro assays. However, golimumab (GLM) has not previously been evaluated. The objective of this study was to compare the activity of CZP with other anti-TNFs, including GLM, in a range of in vitro assays. Materials and methods Neutralisation of human TNF was assessed in the L929 bioassay. The activity of anti-TNFs at inhibiting lipopolysaccharides (LPS)-driven interleukin 1β (IL-1β) secretion by monocytes was measured by ELISA. To determine apoptosis induction, activated peripheral blood lymphocytes and monocytes were examined by annexin V staining using flow cytometry. The effect on neutrophil necrosis was measured by myeloperoxidase release. An isotype-matched control was used in all assays except the L929 bioassay. Results The inhibitory concentration (IC90) neutralisation activity in the L929 bioassay was 0.3 ng/ml for ETA, 4 ng/ml for GLM, 15 ng/ml for ADA and 20 ng/ml for IFX, versus 2.5 ng/ml for CZP. CZP was the most potent inhibitor of LPS-driven IL-1β secretion (IC50 ∼0.1 ng/ml), followed by GLM (20 ng/ml). GLM, ADA, IFX and ETA induced apoptosis of monocytes and lymphocytes to a similar degree. CZP caused no increase in apoptosis above isotype-matched control levels. In the neutrophil necrosis assay, ADA, IFX and GLM caused ∼70% necrosis at 100 μg/ml, and ETA 48%. CZP did not increase the level of necrosis above the level of the control. Conclusion The ability of GLM, IFX and ADA to neutralise human TNF was inferior to CZP and ETA. CZP had a different profile to the other anti-TNFs; it was the most potent at inhibiting monocyte LPS-driven IL-1β production, did not induce apoptosis of activated monocytes and lymphocytes and did not cause neutrophil necrosis. The clinical relevance of these in vitro effects is unknown. Nevertheless, these assays show interesting differences between anti-TNFs.

Mast cells are highly responsive cells found in the skin, which can very rapidly secrete an array of inflammatory mediators. It is possible that severe injection-site pain, which can be seen with some TNF inhibitors, could be linked to the mediators released upon degranulation by mast cells. Certolizumab pegol (CZP) is the only PEGylated Fab' anti-TNF-α for the treatment of Crohn's disease and has been shown to have low levels of injection-site pain in clinical trials.[1] One unique aspect of the structure of CZP is the site-specific attachment of 40 kDa of PEG. The aim of these experiments was to determine the effect of PEG on non-immune-stimulated mast cell degranulation. Mast cells were cultured in vitro from stem cells using the method of Saito et al.[2] over an 8-12 week period. The expression of relevant mast cell markers were tested by flow cytometry. Degranulation, measured by β hexosaminidase release, was stimulated by the addition of the compound 48/80, which is a known non-immune activator of mast cells. Titrations of CZP, PEG and a mixture of PEG and naked Fab' at a PEG concentration of 45 mg/mL were incubated with the mast cells and a fixed amount of compound 48/80 to determine the effect on mast cell degranulation. This is a physiological concentration range at the site of injection, as the CZP is injected at a concentration of 200 mg/mL. The viability of the mast cells at the end of the experiment was assessed using the Promega CellTiter 96 AQueous One Solution Cell Proliferation Assay. The cultured human mast cells expressed markers such as CD117, CD203c and CD32, which are characteristic of tissue mast cells. Compound 48/80 mediated a good level of degranulation, as measured by β hexosaminidase release, although the absolute level varied between cell preparations. CZP, PEG alone and a mixture of naked Fab' and PEG all inhibited the majority of the degranulation in a dose-dependent fashion. No effect on cell viability with any of the reagents tested was seen at the end of the assay. PEG inhibited the degranulation of mast cells stimulated by compound 48/80 at high concentrations. The concentrations at which an effect is observed are what might be expected at the injection site but not systemically. The exact mechanism behind this activity is unclear and needs to be investigated further. This beneficial effect of PEG could explain the low level of injection-site pain observed with CZP in clinical trials.

PEGylated Fab' technology was introduced into the IBD speciality by the approval of certolizumab pegol in the US for CD. All the other anti-TNFs are IgG molecules. Using the collagen II arthritis model, it has been shown that a PEGylated Fab' has a higher inflamed-to-normal tissue ratio of distribution compared to an IgG due to the properties conferred on the molecule by the PEG.[1] PEG has a water shell associated with it that restricts the diffusion of PEGylated proteins out of blood into normal tissue, but not into leaky inflamed tissue. The aim was to examine the biodistribution of a PEGylated Fab' like certolizumab pegol in the intestine in comparison with an IgG. The near IR fluorochrome Alexa Fluor 790 has absorption and emission characteristics that enable it to be used with the IVIS biofluorescence imager. This technology enabled measurement of the level of labeled IgG and PEGylated Fab' in excised colons from animals with established colitis induced by dextran sulfate sodium (DSS). Colitis was induced in Balb/c mice by the addition of 3% w/v DSS in drinking water. Colitis development was assessed by monitoring body weight and signs of diarrhea, and a disease score was assigned to the animals. A PEGylated Fab' and an IgG of an anti-mouse TNF-α antibody (Antibody 501) were labeled with Alexa Fluor 790 following the manufacturer's instructions. Labeling levels were 1.66 molecules of fluorochrome per PEGylated Fab' molecule and 1.1 per IgG molecule. Colitic mice were divided into 2 groups with equivalent disease scores (n= 10/group). 1 group received 0.3 mg sc of labeled PEGylated Fab' and the other group 0.3 mg sc of labeled IgG. Controls were also injected with 0.3mg sc of the labeled PEGylated Fab' or the labeled IgG (n= 8/group). Animals were sacrificed 24 h post-injection and the colons removed. Fluorescence in the normal and inflamed colons was assessed using the IVIS imager. This method allowed accurate estimation of the reagents in the colons of the mice. A region of interest was drawn around the colon to assess the average amount of fluorescence in the tissue. The ratio between the average fluorescence of inflamed vs non-inflamed colons was 3.1 for the PEGylated Fab' and 2.1 for the IgG. This difference between the PEGylated Fab' and IgG was statistically significant (p<0.02) as measured by an unpaired 2 tailed t-test. This method enabled a comparison of the distribution of an anti-TNF-α PEGylated Fab' with an IgG to be assessed in a gut inflammation model. Effective targeting of inflamed tissue is a desired property for an anti-inflammatory agent. The PEGylated Fab' had a higher inflamed-to-normal tissue ratio in the colons than the IgG.

by GW627368X (n= 5) whilst IL1β additions increased Isc by 27.7 ± 9.5 μA.cm-2 and were also abolished by GW627368X (n= 4).We conclude that PGE2, TNFα and IL1β-induced hypersecretion is mediated by EP4 receptors, implicating this receptor as a potential target for the treatment of hypersecretion associated with IBD.

Tumour necrosis factor (TNF)alpha is a proinflammatory cytokine involved in systemic inflammation that mediates chronic inflammatory diseases such as rheumatoid arthritis (RA), Crohn's disease (CD) and psoriasis. Recognition of TNF alpha as a primary mediator of inflammatory disease has driven the development of monoclonal antibodies (mAbs) against TNF alpha as potential novel therapies for these disorders. Certolizumab pegol is a novel, polyethylene glycol (PEG)-conjugated, humanised, antigen-binding fragment (Fab') of an anti-TNF alpha mAb that does not mediate apoptosis or neutrophil degranulation. Preclinical studies have shown excellent bioavailability, with preferential distribution and retention in inflamed tissue, which could be due to the low diffusion rate of PEGylated molecules and/or the lack of an Fc, which prevents FcRn-mediated transport. Pharmacokinetics are linear and predictable. Certolizumab pegol is a potentially valuable new treatment option for several inflammatory diseases. It has shown promising efficacy and tolerability results in Phase II and III trials for RA, CD and psoriasis.

The mechanisms remain uncertain but may involve the cleavage of the receptor which is TNF sensitive.The predictive value of baseline IL15 with respect to therapeutic response may be of practical interest provided confirmation by prospective studies a P < 0.05 versus controls, b P < 0.05 versus week 0, c P < 0.05 versus responders, d four patients.

Purpose: Purpose: Certolizumab pegol, adalimumab and infliximab have been shown to dramatically inhibit the LPS-stimulated production of inflammatory cytokines such as IL-1β by monocytes, whereas etanercept is considerably less potent at mediating this effect. 1 This function is thought to be initiated by signaling through membrane TNFα, although the exact mechanism is not completely understood. The aim of this study was to examine the effect of these anti-TNFα reagents on the levels of cell surface molecules such as Toll Like Receptor (TLR) 2 (CD282), TLR4 (CD284) and CD14, which are involved in the response to LPS. TLR2 and 4 levels have been shown to be upregulated during inflammation, particularly in Crohn's disease. Methods: Heparinised whole blood from healthy volunteers was incubated for 1 hour at 37°C with 10 μg/mL of 1 of the 4 anti-TNFα reagents to allow signaling through membrane TNF. Then 100 ng/mL LPS was added for 2 hours. The cells were stained with either phycoerythrin labeled anti-TLR2, -TLR4 or -CD14 antibody, and a fluorescein isothiocyanate labeled CD33 antibody. Erythrocytes were removed by hypotonic lysis and the samples were analysed by flow cytometry. The level of staining on monocytes was determined by gating the cells using the CD33 antibody staining and side scatter. Results: Certolizumab pegol, adalimumab and infliximab reduced the geometric mean level of TLR2 to 12.2, 18.1 and 14.6, respectively (LPS control was 50.6), TLR4 to 6.1 5.8 and 5.0, respectively (LPS control was 13.2), and CD14 to 47.0, 66.9 and 48.2, respectively (LPS control was 107). Etanercept reduced, to a lesser degree, the level of these 3 surface markers to 37.9, 11.1 and 60.6 for TLR2, TLR4 and CD14, respectively. Conclusion: Certolizumab pegol, adalimumab and infliximab, which have been shown to potently inhibit the LPS driven cytokine production by monocytes, all dramatically reduced the level of TLR2, 4 and CD14 on the cell surface of monocytes activated with LPS. This reduction in the levels of the surface markers could explain the unresponsiveness to LPS as there are less of these proteins on the cell surface to bind LPS and transfer the inflammatory signal into the cell. Etanercept did not reduce the levels of TLR2 and 4 to the same degree and does not potently inhibit the LPS-driven cytokine production. This mechanism of action could be important in Crohn's disease where bacteria are a major part of the inflammatory process.

Purpose: Certolizumab pegol, an Fc-free PEGylated Fab’, binds human tumor necrosis factor (TNF) α with high affinity. The PEG component of certolizumab pegol comprises two 20 kDa chains of PEG-monomethyl ether attached covalently and site-specifically to a hinge thiol on the Fab’. Hemodynamic properties of proteins are altered by PEGylation, leading to preferential penetration into diseased versus normal tissue.1 In this study we monitored metabolism of PEG in rats following subcutaneous administration of certolizumab pegol. Methods: Subcutaneous injections of certolizumab pegol 400 mg/kg were given to nine female Lewis rats. The rats were housed in groups of three in metabolism cages for four 1-week periods (total study duration: 84 days). Daily urine and feces collections were weighed, and stored at −70°C until analysis. PEG levels were quantified using proton nuclear magnetic resonance (1H NMR) in urine samples purified by ultrafiltration. Results: The mean recovery of PEG from urine and feces combined was 91% of the dose. PEG was detected in all urine samples; concentrations reached a maximum (198 μg/mL) on Day 4, and then declined in a first-order manner to 14 μg/mL in Week 12. The mean daily excretion in urine (as a proportion of the dose) was maximal (1.9%) on Day 6, declining in a first-order manner to 0.21% per day in Week 12. After 84 days, the mean cumulative amount of dose excreted in urine was 65%. Using a first-order increase/decrease model, the total urinary excretion was 73% and the half-life for the decrease in daily urinary excretion was 23 days. The molecular weight of the PEG excreted in urine was estimated by SDS PAGE to be 40 kDa. Up to Day 42, PEG equivalent to 18% of the dose was detected in feces (much of the fecal PEG is thought to be a result of urine contamination). Conclusion: The innovative method of 1H NMR spectroscopy shows that, in the rat, the 40 kDa PEG moiety of certolizumab pegol is cleaved from the Fab’ and excreted unchanged in a first-order process, predominantly in the urine. The half-life for excretion of a single dose determines the time taken to reach steady-state excretion on multiple dosing. The rate of excretion during multiple dosing matched the rate of dosing at steady-state as cumulative excretion was near-quantitative. This research was funded by UCB.